The intervertebral discs lie between adjacent vertebrae bodies in the spine. Each disc forms a cartilaginous joint that allows slight movements of the vertebrae, acts as a ligament that holds the vertebrae together, and supports compressive loads arising from body weight and muscle tension.
Cartilage is a firm, resilient connective tissue composed of specialized cells called chondrocytes that produce a large amount of extra cellular matrix (ECM). It provides protective cushioning and enables the joints to withstand loads arising from motion needed to perform every-day activities. The body contains three different types of cartilage: articular, which covers joint surfaces; fibro cartilage, which is found in the knee meniscus and intervertebral disc; and elastic cartilage, which is found in the outer ear. The different cartilages are distinguished by their structure, elasticity, and strength.
Aota et al (“Differential effects of fibronectin fragment on proteoglycan metabolism by intervertebral disc cells: a comparison with articular chondrocytes”. Spine. 2005; 30:722-728) reported on different effects of fibronectin fragment on proliferation and proteoglycan metabolism in different populations of intervertebral disc and articular chondrocytes. These results suggested that chondrocytes from different cartilage tissue require different conditions for proliferating and for maintaining tissue function.
The intervertebral disc is composed of three basic structures: an inner gel-like substance called the nucleus pulposus (NP), a tough fibrous outer band called the annulus fibrosus (AF), and superior and inferior cartilaginous endplates, which mark the transition between the intervertebral disc and the vertebra. These structures differ in the arrangement of proteoglycan and collagen in the tissue, as well as the relative concentration of each.
During embryonic development three germ layers can be differentiated: the endoderm, the mesoderm, and the ectoderm. These three layers ultimately give rise to internal organs; musculoskeletal tissues; and epidermal and nervous tissues, respectively. A fourth region, known as the notochord, guides the embryonic development of the neural tube; and the vertebral column, including the intervertebral discs. Mesenchymal cells begin to migrate and condense around the notochord to form the osseous vertebral bodies and the annulus fibrosus. The entrapped notochordal cells play a critical role in initiating the development of the nucleus pulposus (Walmsley R. “The development and growth of the intervertebral disc”. Edinburgh Med J. 1953; 60:341-364). Thus, these two basic structures of the intervertebral disc, the annulus fibrosus and the nucleus pulposus, are originated from different embryonic origin namely the mesenchyme and the notochord, respectively.
The annulus fibrosus is primarily composed of type I collagen fibrils that form concentric lamellae that surround the nucleus pulposus and insert into the endplates of the adjacent vertebral bodies to form a reinforced structure.
The nucleus pulposus consists of predominantly small chondrocytes-like cells and a second population of large highly vacuolated cells these are the notochordal cells which are presumed remnants of the embryonic tissue that guided formation of the spine and the nuclei pulposi (Hunter et al, “The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering”. Tissue Eng. 2003; 9:667-677). Due to the fact that these notochordal cells appear to play a crucial role in formation of the spine and the nucleus pulposus, it is presumed that they could promote repair of the damaged disc and spine. The chondrocytes-like cells express type II collagen, proteoglycan aggrecans, and hyaluronan long chains, which have molecules with highly hydrophilic, branching side chains. These negatively charged regions have a strong avidity for water molecules and hydrate the nucleus or center of the disc by an osmotic swelling pressure. The hydraulic effect of the contained hydrated nucleus within the annulus acts as a shock absorber to cushion the spinal column from forces that are applied to the musculoskeletal system. The vertebral endplates are attached to both the disc and the adjacent vertebral body. The chemical structure of these plates consists of proteoglycan and collagen fibers.
Human intervertebral disc degeneration is a clinical problem, and leading cause of spinal pain and disability. Over 15 million people worldwide suffer from disc degeneration, which is typically characterized by having an altered matrix composition and reduced cell number.
Degenerative Disc Disease (DDD) is an undesired process in which the intervertebral discs lose their flexibility, elasticity, and shock absorbing characteristics. In this process the collagen structure of the annulus fibrosus weakens and becomes brittle. Excessive pressure on a weakened disc can cause tears in the annulus fibrosus, enabling the nucleus pulposus to herniate or extrude through the tears this condition is called herniated disc. The herniated material can compress the nerves around the disc and create pain. A herniated disc can interfere with nerve function, leading to weakness, numbness, inflammation and pain. Additionally, proteoglycan content decreases, thereby reducing the water retaining capabilities of the nucleus pulposus. These changes reduce the ability of the discs to act as shock absorbers and make them less flexible. Loss of fluid also makes the disc smaller and narrows the distance between the vertebrae and the disc.
Since the intervertebral discs are located in a non-vascular environment, the use of tissue engineering of a disc to slow or reverse the degenerative process represents a major biological challenge as they have a limited capacity for repair. Cell activity requires glucose, oxygen, and other nutrients necessary for tissue supporting. However, the disc is the largest avascular tissue in the body. The cells within the disc are sustained by diffusion of nutrients into the disc through the porous central concavity of the vertebral endplate (Rudert M and Tillmann B. “Lymph and blood supply of the human intervertebral disc. Cadaver study of correlations to discitis”. Acta Orthop Scand. 1993; 64:37-40).
Degeneration of intervertebral discs or a hematoma can cause spinal cord compression and spinal injury. Spinal cord injury (SCI) usually begins with trauma to the spine that damages the nerves within the spinal canal. Frequent causes of damage are trauma (car accident, gunshot, falls, etc.) or disease (e.g. polio, spina bifida, Friedreich's Ataxia, etc.) and compression of the spine by intervertebral disc herniation. An injury to the spinal cord nerves results in loss or deficit in motor, sensory, and autonomic function. Secondary injury following the primary impact includes a number of biochemical and cellular alterations leading to tissue necrosis and cell death. It is estimated that the annual incidence of SCI is approximately 40 cases per million in the U.S., and that the cost of managing the care of SCI patients approaches $4 billion each year. To date, relatively little progress has been made in the treatment of SCI and related neurological impairments. Currently there is only one accepted, although unapproved, therapy, methylprednisolone. If used in very high doses no later than 8 hours after the injury, methlyprednisolone has demonstrated a modest ability to improve the neurological outcome following SCI.
In the developing vertebrate nervous system, the neural tube is the precursor of the central nervous system (CNS), which comprises the brain and spinal cord. The spinal cord contains communication fibers called axons that transfer sensory and motor information between the brain and the periphery. In transverse section the spinal cord is divided into symmetrical halves by a dorsal median and a ventral median. The dorsal part of the neural tube is primarily associated with sensation, whereas the ventral part is primarily associated with motor (e.g. muscle) control. The term motor neuron classically applies to neurons located in the CNS which project their axons outside the CNS and directly or indirectly control muscles. The term is synonymous with efferent neurons. An injury to the spinal cord has devastating implications resulting in loss of sensation or motor function below the injury level. Following an injury to the CNS, motor neurons are unable to re-grow their axons and they die by necrosis or apoptosis. Published research shows that by transplanting and expressing a second notochord near the dorsal neural tube, 180 degrees opposite of the normal notochord location, one can induce the formation of motor neurons in the dorsal tube, which generally forms sensory cells. An injured CNS is a highly inhibitory environment for axon regeneration, severely limiting functional recovery following injury.
Fibrin glue is typically a blood product obtained from either commercial sources or some regional blood transfusion centers. Components that are commonly used in the preparation of fibrin glue are fibrinogen, thrombin, Factor VIII, Factor XIII, fibronectin, vitronectin and von Willebrand factor (vWF). Fibrin glue formulations are used in surgery, both as a useful addition to sutures and to provide optimal wound integrity, for haemostasis, and for preventing or treating adhesions. Some manufacturers add anti-proteolytic agents to the fibrin glue formulation (as described in WO-A-93/05822) or specifically remove the plasminogen in order to delay or stop the fibrinolysis (as described in U.S. Pat. Nos. 5,792,835 and 7,125,569).
Typically, cryoprecipitation preparation from plasma is the first step in the manufacture of fibrinogen of fibrin based adhesive. Bar et al (“The binding of fibrin sealant to collagen is influenced by the method of purification and the cross-linked fibrinogen-fibronectin (heteronectin) content of the ‘fibrinogen’ component”. Blood Coagul Fibrinolysis. 2005; 16:111-117) reported that fibrin gel formulations prepared from cryoprecipitate differ in their content of fibronectin and heteronectin (fibrinogen-fibronectin covalently linked complexes). The report indicated that content of heteronectin in the formulation influences the fibrin based adhesion to collagen. On the one hand, Schwartz et al (U.S. Pat. No. 4,377,572) purification procedure results in removal of most of the cross linked fibrinogen-fibronectin molecules, subsequently resulting in a low fibronectin:fibrinogen ratio of 1/14.7 in the formulation and low collagen and gelatin-binding properties of the formed fibrin. On the other hand, the cryoprecipitate preparation described by Martinowitz and Bal (EP-B-691,858) preserves these cross linked fibrinogen-fibronectin molecules, and consequently has an increased fibronectin/fibrinogen ratio of 1/7 which correlates with increased adherence of the produced fibrin to collagen as compared to adherence to collagen of the fibrin formed with the preparation obtained by the Schwartz purification.
The following publications disclose the use of different fibrin glue preparations in spinal disease. Three-dimensional fibrin matrices as cellular substrates in vitro and as bridging materials for central nervous system repair have been reported. Ju et al (“Enhanced neurite growth from mammalian neurons in three-dimensional salmon fibrin gels. Biomaterials”. 2007; 28:2097-2108) reported that salmon fibrin gels were superior scaffold for neuronal re-growth after CNS injury as compared to fibrin prepared from human or bovine blood proteins. Cheng et al (“Spinal cord repair in adult paraplegic rats: partial restoration of hind limb function”. Science. 1996; 273:510-513) describes repair of spinal cord gaps in adult rats using peripheral nerve grafts. The grafted area was stabilized with fibrin glue containing acidic fibroblast growth factor.
US patent application US-A-2004/0121011 describes a method for promoting repair, regeneration and re-growth of injured neuronal cells. The application indicated that the nerve injury site can be in the central or in the peripheral nervous system. The formulation combines Rho antagonist and a flowable carrier component capable of forming an acceptable matrix in vivo such as tissue adhesives. US-A-2004/0121011 discloses different protein-based tissue adhesives including collagen gels, fibrin tissue adhesives, matrigel, laminin networks, and adhesives based on a composition of basment membrane proteins that contain collagen. Various commercial preparations are disclosed such as, Tissucol®/TISSEEL®, Beriplast® P, and Hemaseel®.
The following publications disclose the use of fibrin glue compositions in intervertebral disc.
US-A-2005/0148512 relates to injection of a fibrosing agent or a composition comprising a fibrosing agent into damaged intervertebral discs to enhance scarring and support the annular ring of the disc. Fibrinogen-containing formulations such as TISSEAL® are mentioned among numerous compositions which can be delivered into the intervertebral disc.
U.S. Pat. No. 6,428,576 describes a method for repairing defects in the annulus fibrosus using an in-situ curing sealant. The patent discloses a formulation that cure to a viscoelastic material that simulates the structure, physical properties and biomechanical functions of the annulus fibrosus. The cured polymer may be synthetic or naturally occurring. The patent discloses that synthetic polymers are more reliable. The patent discloses several natural occurring proteins, such as albumin, collagen, fibrinogen, fibrin and elastin. These proteins can be from any source such as protein fractionated from blood or recombinant proteins, including processed, denatured or otherwise modified.
WO-A-07/089942 discloses a method of treating a disc, comprising injecting a fibrin sealant into a disc to seal at least one defect of an annulus fibrosus while monitoring the pressure of the fibrin sealant being injected. The fibrin sealant comprises fibrinogen and an activating compound such as thrombin. According to the description the defect can be a tear or a fissure in the annulus fibrosus or a fibrous capsule of a spinal joint. The description discloses use of any fibrinogen that will form fibrin in a human body. Fibronectin is mentioned as one of numerous possible additives to the fibrinogen composition.
WO-A-06/050268 discloses an injection of fibrin sealant into a tear or a fissure in the annulus fibrosus. The sealant comprises fibrinogen and activating compound such as thrombin. According to the application, the fibrinogen component can be autologous, human including pooled human fibrinogen, recombinant, and bovine or other non-human source. Fibronectin is mentioned among many components as an additional additive which can be employed in the fibrin sealant.
Also WO-A-06/050267 discloses the injection of fibrin sealant and anesthetic into the spinal area to seal defects in the annulus fibrosus, such as tears or fissures. According to the description, the fibrinogen component includes any fibrinogen that will form fibrin in human body. The application mentions commercial kits from manufacturers as Baxter such as TISSEEL®. The description of WO-A-06/050267 discloses that alternative amounts of fibrinogen may be used in order to change the density of the combined components.
WO-A-07/089948 relates to a method of treating a disc that is leaking nucleus pulposus into and/or thorough at least one defect in the annulus fibrosus. The method comprises injecting a biological sealant, such as fibrinogen solution and activating solution to the spinal area using a multi-lumen catheter. The description also relates to a kit comprising a biological sealant and a biological sealant apparatus for injecting fibrin sealant into a human disc.
U.S. Pat. No. 6,468,527 describes a two component fibrin sealant including a biological or non-biological agent. The composition provides a mean for delivering a particular agent to a specific critical site within the body and providing a prolonged, time release therapeutic value. Injections of fibrin glue infused with corticosteroids into the lumbar epidural space and into the intra discal space are specifically disclosed. The fibrin sealant acts to maintain extended anti-inflammatory response of the corticosteroid and to seal the annular fissures, which otherwise allow damaging chemicals to escape from the disc space and bathe the nerve root resulting in chemical radiculitis. Also, U.S. Pat. No. 7,235,255 discloses a system for delivering a biological tissue adhesive comprising a fibrinogen component, a thrombin component, and a corticosteroid-containing solution. According to the description the fibrin sealant can be used to treat degenerative disc and incompetent disc disease. Exemplified is an intra-discal injection. The delivery system seals, protects the exposed nerve roots from further chemical damage, and acts as a vehicle to maintain corticosteroids in a lasting deposition on the nerve root.
The following publications report tissue engineering as a possible biologic approach, which aims to replace, repair, maintain, and/or enhance tissue function by combination of cells, suitable biochemical and physiochemical factors and optionally a porous structure to be employed as scaffold. WO-A-04/093934 discloses a method of augmenting and/or repairing an intervertebral disc by administering stem cell material into the disc. The stem cell material is provided in a biologically compatible lattice material. The preferred lattice material is lipo-derived lattice such as proteoglycans, glycoproteins, hyaluronins, fibronectins, collagens (type I, type II, type III, type IV, type V, type VI, etc.), and the like. According to the description of WO-A-04/093934 the lipo-derived lattices serve as excellent substrates for cell growth. Exemplified are only collagen-based lattice materials.
WO-A-00/47621 discloses a method for producing a viral inactivated cryoprecipitate having a preferred fibrinogen and fibronectin ratio of from 0.02 to 0.5 which e.g. can be used to produce a fibrin based biomatrix suitable for growing any human cells and keratinocytes, fibroblasts and chondrocytes are mentioned. In one preferred embodiment, the antifibrinolytic agent t-AMCHA (i.e. tranexamic acid) which is indicated to advantageously lower the viscosity of the composition is used.
U.S. patent application Ser. No.06/0275273, describes a method for implantation or injection of chondrocytes into a degenerative intervertebral disc. The patent discloses chondrocytes obtained from cadaver. According to the description the chondrocytes can be obtained from cartilage tissue, including intervertebral disc cartilage, or cartilage originating from cartilaginous tissues other than intervertebral disc tissue. The description discloses several biocompatible molecules to be added to the cell composition such as laminin, chitosan, hydrogel, pegylated hydrogel, collagen type I, II, III, fibrinogen, fibrin, thrombin, fibronectin and hyaluronic acid. Disclosed is the use of commercial formulation TISSEEL® fibrin glue with the cells. The examples also disclose the use of cryoprecipitated porcine fibrinogen and a chondrocyte-thrombin solution.
U.S. patent application Ser. No.07/0093905 discloses a mixture for repair and regeneration of intervertebral discs comprising glycine, concentrated monocytes and fibrin glue. The patent also discloses excised and treated nucleus or annular tissue for reinsertion into the disc. The reinserted disc cells can optionally be combined with carriers such as a gel-like carrier or an adhesive. The gel like carrier can be a biological or synthetic hydrogel, hyaluronic acid, collagen gel, mussel-based adhesive, fibrin glue, fibrin clot, blood, blood clot, blood component, blood component clot etc. The patent application does not mention a specific composition of the disclosed carriers, and is silent on a cryoprecipitate concentrate.
EP-A-1,894,581 discloses a matrix gel comprising chondrocytes or progenitor cells as a cartilage repair implant. According to the description the gel matrix provides a simple dilution of primary chondrocytes resulting in increased production of extra cellular matrix material. In a preferred embodiment the chondrocytes are isolated from articular cartilage. Fibrin glue is mentioned among numerous matrix gel material that can be used. The application does not mention a specific composition of the matrix gel material and is silent on a particular relative concentration among the gel components.
Disc cells grown in monolayer assume a fibroblast-like phenotype. In a three-dimensional environment, however, disc cells become rounded, form colonies, and exhibit greater proliferation and proteoglycan synthesis. Various in vitro culture techniques, including complex three-dimensional gels and degradable polymer scaffolds have been developed with the goal of providing a sustainable frame on which the disc cells can proliferate. Hyaluronic acid, collagen, chitosan and fibrin gel have been used in cross-linkable polymeric preparation to entrap cells.
In spite of all the techniques reported on the use of fibrin glue in IVD, Gruber et al (“Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers”. Spine J. 2004; 4:44-55) reported that fibrin gel formulations were inferior microenvironments for proliferation, ECM production and gene expression of annulus fibrosus cells. The term “extra cellular matrix”, abbreviated “ECM”, refers to the complex structural material that is produced by cells in mammalian tissues. The extra cellular matrix is typically the defining feature of a connective tissue, for example, chondrocyte cells. The ECM in vivo usually provides structural support to the cells.
There is a need for an optimal fibrin composition suitable for treating a spine disease, disorder or condition such as intervertebral disc degeneration.